© 2021 Wiley Periodicals LLC.This study was carried out to reveal the relationship of the brain with both the mandibular lymph node (MLN) and parotid lymph node (PLN) by the hyperspectral fluorescence imaging techniques of Qdot 800 (QD) nanoparticles using in vivo. This relationship of the brain with both lymph nodes offers the preliminary morphological definition of lymphatic drainage. QD was injected into the left parietal brain lobe of each rat at a depth of 2.50 mm. In 65% of the rats that were imaged in vivo, signals were received first from the right MLN and PLN, and then from the left MLN and PLN. In contrast, in two female rats, the first signal was received from the right PLN. There was no difference between the female and male rats overall. The most noteworthy finding of this study was that the tracer injected into the left parietal lobe reached the right mandibular and parotid lymph nodules earlier. This result indicates a different and unknown pathway in the brain that communicates with the lymph nodes. Moreover, this study shows that these lymph nodes pathways can be used in the treatment of diseases such as brain trauma, cerebral edema, and Alzheimer's disease (AD).